The overall goal is to evaluate the role of reactive oxygen intermediates (ROl) in the molecular mechanisms by which ethanol is toxic. Major focus will be on the role of P4502E1, interaction with iron, and reactivity of NADH and NADPH as cofactors. Specific Aim 1 - Our laboratory developed a HepG2 cell line which stably expresses human P4502E1. Ethanol was toxic to these cells, but not to control cells. These cells appear to represent a novel model to assess the role of P4502E1 or ROl generated during the metabolism of ethanol by P4502E1 in the hepatotoxic actions of ethanol. Studies will be carried out to characterize ethanol toxicity, effect of P4502E1 inhibitors, antioxidants, iron chelators, enrichment of cell membranes with PUFA and the ability of ethanol, acting via P4502E1,to produce a state of oxidative stress: Lipid, protein, and DNA targets for ROl produced by ethanol will be studied. Acetaldehyde and lipid adducts will be immunochemically detected. A final study will be to assess whether ethanol toxicity involves an apoptotic mechanism; if so, protection by BCl-2 will be studied. Specific Aim 2 - The role of NADH-cyt b5 reductase, cyt b5, and cyt P450 in the NADH-dependent production of ROl by microsomes will be studied employing reconstituted systems containing the purified enzymes, and inhibitors and antibodies against these enzymes. Specific Aim 3 - The interaction of NADPH-P450 reductase and P4502E1 with various ferric complexes to produce ROl will be investigated to test the hypothesis that the ability of the reductase versus P450 to interact with iron is not only dependent upon the chelated form of the iron, but also the concentration of iron. Specific Aim 4 - The production of ROl by microsomes and mitochondria isolated from periportal and pericentral hepatocytes of control and ethanol-treated rats will be determined since P4502E1 is present at highest levels in the PC zone and ethanol injury originates here. Specific Aim 5 - Studies will be carried out to compare the production of ROl by human P4502E1 with other human P450 isozymes. Ethanol toxicity and the role of 2E1 and ROl will be studied in immortalized normal human liver cells. These studies will help define the role of P4502E1 and ROl in the mechanism by which ethanol is hepatotoxic, help to design therapeutic interventions to prevent or ameliorate this toxicity, and provide basic mechanistic information of value to the oxygen radical and P450 areas of research.